The present invention relates to high-throughput methods and apparatus for ultra-sensitive biological assays with sufficient sensitivity to detect and identify single biomolecules. More particularly this invention relates to the detection of single target biomolecules through the use of two or more probe molecules each labeled with distinctive optical tags (such as fluorescent molecules, plasmon resonant particles, or quantum dots) that, selectively and specifically, recognize and form bound complexes with the target. Present immuno-, protein, or DNA (without amplification by polymerase chain reaction (PCR) or related techniques) assays have sensitivity thresholds in the nanomolar to picomolar range. Assays performed using the present invention are capable of extending the threshold of detection to femtomolar or lower concentrations with practical measurement times of 1–100 minutes.
Present immuno-and DNA assays (without amplification) have sensitivity thresholds in the nanomolar to picomolar range. Researchers have attempted to improve the sensitivity by several orders of magnitude by employing newly developed single molecule optical microscopy detection methods. These attempts have been partially successful but at the moment appear to be limited by non-specific surface attachment of unwanted labels or by impractically long measurement times.
Considering previous work, Mathies et al, U.S. Pat. No. 4,979,824 issued Dec. 25, 1990, describes how to detect single fluorescent particles and molecules with a fluid flow stream. They used only single-probe fluorescent labels, which suffers from the following shortcoming. A washing step is required to remove all of the unbound probe molecules from the sample. If some of the unbound reporter remains in the sample container, they can be mistaken for the presence of the target molecules and thus give a false positive result. As described here, the rate of false positives can be dramatically reduced by the use of two probe molecules, each with its own distinctive label.
Mathies et al also demonstrates the use of a fluid flow stream to carry sample molecules through the interrogating region of the optical detection system. They however use a fluid geometry that is either a small diameter stream of fluid that has limited capability for moving a significant sample volume through the interrogation region in a reasonable time period, or they use a 2D fluid sheet in which the sample volume is interrogated by scanning the focal volume in the x and y directions across the sample. Again the length of time to perform this type of assay can be impractically long.
Trabesinge et al, Anal. Chem. 2001, 73, 1100–1105, point out the advantages of two-colors in their assays but do not employ a high throughput device for examining practical sample volumes in practical time periods. Additionally they do not use dual probes in their assay methods.
Ma et al, Anal. Chem. 2000, 72, 4640–4645; Lyon et al, Anal. Chem. 1997, 69, 3400–3405; and Zander et al, Chemical Physics Letters 286 (1998) 457–465, use two-colors with dual probes in a flow system but they use only a 1-D small diameter flow stream and do not achieve the same degree of throughput as can be achieved with the 2D flow stream and image detection described here.
Loscher et al, Anal Chem. 1998, 70, 3202–3205; and Erkisson et al, WO99/40416, Aug. 12, 1999, describe the use of single molecule detection for ultra sensitive assays but do not describe the use of dual probes or high throughput methods.
The prior art does not provide a method and apparatus for performing high throughput ultrasensitive bioassays at the single molecule level with low false positive backgrounds.
This invention applies to immunoassays where the target biomolecule is an antigen. The assay uses at least two specific antibodies that act as probes by strongly binding to the target antigen without binding to other biomolecules. The assay then consists of labeling the two (for instance) antibodies with distinctive optical tags that can then be individually observed with a sensitive optical detection method. If the detection method reveals that the two antibodies are spatially co-located within the resolution of the optical imaging system and the frequency of co-localization events is greater than expected from random coincidence, the assay is positive for the presence target antigen and allows for a quantitative measure of its concentration.
We describe two optical detection methods for determining co-location of two or more optically labeled probe molecules. The first is an apparatus and method in which target antigens are captured by an antibody that has previously been attached to a solid surface substrate. The presence of the labeled antibodies is determined by scanned or wide-field imaging of the substrate. A positive result for the presence of the antigen is then given by the binding of two additional monoclonal antibodies, each having a distinct optical label. The number of co-localized events must be above background number of accidental co-located optical labels. This method is limited in sensitivity by non-specific surface binding by the labeled antibodies.
The second apparatus and method uses a 2D flowing fluid sheet confined by a microfluidic channel. This apparatus also uses the co-location of two or more optical labels but is not limited by non-specific surface attachment of antibodies because the targets and antibodies are primarily entrained in the moving fluid and are not in contact with the surfaces. This method gives 103–104 times greater throughput than previous 1D micro-stream devices (Mathies et. al.). Using this method, immunoassays at ultra-low (femtomolar and lower) concentrations can be performed in short time periods (10's of seconds to 10's of minutes).
In addition to immunoassays, these methods and apparatus can also be applied to assays of short (10–50 bases) DNA or RNA oligonucliotides having specific target sequences. Here two or more single strand DNA complements each having distinct optical labels are used to specifically bind to the single strand target DNA fragment. Yet, Another class of assays is the detection of specific proteins or protein complexes. Here two or more labeled proteins or small molecules that bind to the target biomolecule are used as probes.
The invention described here is able to perform high throughput binding assays at the single molecule detection limit with low background by using the following features:                a) Dual probes—This reduces the background due to non-specific binding of the probe molecules        b) 2 or more colors—Each probe is labeled with a spectrally distinct optical label        c) This assay does not require the target molecule be directly labeled. Its presence is determined by the detection of labeled affinity probes.        d) Large interrogation volumes are employed to facilitate high-throughput. This is accomplished by using wide-field video rate, amplified CCD imaging, total internal reflection and microfluidic 2D flow-channel fluid manipulation.        
The term “probes” as used herein is defined as antibodies, oligonucleolides, or proteins, for example.